1) Molecular machinery regulating protein secretion in the acinar cells of salivary glands The major secretory units in the salivary glands are the acini that are formed by polarized cells, which form a small lumen (acinar canaliculi) where salivary proteins and water are secreted. Proteins destined to secretion are synthesized in the endoplasmic reticulum, transported through the Golgi apparatus to the trans-Golgi network (TGN), packed in secretory granules and finally released into the cytoplasm and transported to the cell periphery. Here, upon stimulation of G protein coupled receptors, the granules fuse with the apical domain of the plasma membrane (PM) releasing their content into the apical lumen. Our goal is to study the molecular machinery regulating the formation of the granules at the TGN and their fusion with the PM in live animals. To this aim, we have been using a transgenic mouse model ubiquitously expressing the soluble green fluorescent protein (GFP). The advantage of this model system is that the secretory granules and the apical domain in the acini can be easily imaged during stimulated secretion. We have found that in both submandibular and parotid glands, stimulation of the adrenergic, but not the muscarinic receptors, is the major trigger of the pathway leading to the exocytosis of the granules. Furthermore, by following the dynamics of single granules we have found that adrenergic stimulation enhances the mobility of the secretory granules that are delivered to the apical pole of the plasma membrane, where they dock, recruit actin and finally fuse in a single step, releasing their content into the acinar canaliculi. 2) Molecular machinery regulating the sorting of proteins from the TGN to the apical or the basolateral domain of the plasma membrane Proteins are sorted from the TGN to the apical or the basolateral domain of the PM. Whether these proteins follow a direct route to the PM or they are first diverted towards other intracellular compartments is still a matter of debate. Furthermore, the molecular machineries regulating the sorting events are not still understood. We are focusing our attention on two proteins belonging to a family of water channels expressed in acinar cells, aquaporin 3 (AQP3) and aquaporin 5 (AQP5), which are localized to the basolateral and the apical domain respectively. Our strategy is to transduce in live animals fluorescently tagged AQP3 and AQP5 and to assess by time lapse imaging the route followed by these proteins once they are released from the TGN. Since preliminary results suggest that the sorting signals are located at the C-terminus of AQP3 and at the N-terminus of AQP5, we are going to introduce mutations in these domains in order to identify the sorting motifs. Lastly, we will use these domains as baits to identify molecular components of the sorting machinery at the TGN by either 2-hybrid screening or biochemical assays. Establishing an experimental procedure to rapidly express genes in the salivary glands We have set up an experimental procedure to rapidly and effectively transduce various genes in to the salivary glands of live rodents by using plasmid DNA. Specifically we have shown that under different experimental conditions, a reporter molecule can be rapidly expressed in specific compartments of the glands: i) in the intercalated ducts, when plasmid DNA is administered alone, and ii) in granular ducts, striated ducts and to a lesser extent in acini, when plasmid DNA is mixed with replication deficient adenovirus subtype 5 (rAd5) particles. Remarkably, we also found that gene expression can be directed to acinar cells, when plasmid DNA is administered while exocytosis is being stimulated with isoproterenol, which promotes saliva secretion, suggesting a novel mechanism of plasmid internalization regulated by compensatory endocytosis. Finally, we have show that these procedures can be utilized to express any fluorescently tagged molecule enabling the study of the dynamics of various cellular events. Expression and localization of AQP5 and AQP3 in the acini of the salivary glands We have cloned AQP3 and AQP5 from both mice and rats and tagged them with various fluorescent proteins including YFP, CFP, mCherry and the photo-activatable variants of GFP and RFP. These constructs were expressed in various cell lines such as HSG and HSY grown in matrigel, and MDCKI and II. The tagged proteins localize both at the plasma membrane and in some intracellular compartments and analysis by western blotting shows that the proteins are properly processed. Next, we have expressed these proteins in the acinar cells of SMGs of live rats. Notably, fluorescently tagged AQP5 localized at the apical domain of the acinar cells and in a subset of intracellular vesicles, as shown for the endogenous proteins. Furthermore we have imaged dynamically the movement of the intracellular vesicles in the live animals co-expressing a marker for the TGN. Fluorescently tagged AQP3 did not localize at the apical pole of the plasma membrane but was rather observed in a series of intracellular compartments that were not yet characterized. Finally, we have expressed and set up the conditions to express and image the photo-activatable variants of AQP5 and AQP3 together with a fluorescently tagged marker of the TGN, namely TGN-38. 3) Molecular machinery regulating endocytosis in salivary glands of live animals The role of endocytosis in the physiology of the salivary glands has never been explored. Uptake of proteins from the apical domain of the ductal system and uptake of proteins from the basolateral domain have been described but never thoroughly investigated. The presence under physiological conditions of salivary proteins in the bloodstream and of serum proteins in the saliva, argues strongly in favor of a constant and bi-directional transcytotic movement of proteins across the salivary gland epithelium. The salivary glands offer a unique opportunity to study endocytosis in polarized epithelial cells in a physiological context since both the basolateral and the apical domains are accessible from the blood stream and from the major excretory duct, respectively. Our aim is to define the endocytic pathways in the salivary glands, to investigate the molecular machinery regulating the internalization of various cargo with a particular emphasis on the role of the cytoskeleton, and finally to understand what is the contribution of the endocytic events in the physiology of the glands and especially during secretion. We set up an experimental system to study endocytosis in live rodents by intravital TPM. First, we showed that by taking advantage of the unique properties of the TPM, the architecture of the salivary glands can be visualized in great detail analyzing at the same time the parenchyma of the tissue, the extracellular matrix, and a broad spectrum of fluorescent probes that are administered exogenously. Next we showed that systemically injected probes such as dextrans, bovine serum albumin (BSA), and transferrin (Tfn)can be internalized primarily by the fibroblasts present in the stroma of the glands. We showed that the internalization of these probes occurs within the first few minutes and that their trafficking from the early to late endosomal/lysosomal compartments can be followed dynamically in the live animal at a resolution comparable to that achieved in cell culture by confocal microscopy. Strikingly we also observed that dextran was internalized with a much faster kinetic than Tfn in live animals. When fibroblasts were isolated and cultured on solid surfaces, Tfn was internalized within 30 sec while dextran appeared internalized after 20-30 minutes. These results strongly indicate that cell culture systems might behave very differently than cell in their physiological environment